Magnetic nanoparticles enhance the cellular immune response of dendritic cell tumor vaccines by realizing the cytoplasmic delivery of tumor antigens

Abstract Dendritic cells (DCs)‐based tumor vaccines have the advantages of high safety and rapid activation of T cells, and have been approved for clinical tumor treatment. However, the conventional DC vaccines have some severe problems, such as poor activation of DCs in vitro, low level of antigen presentation, reduced cell viability, and difficulty in targeting lymph nodes in vivo, resulting in poor clinical therapeutic effects. In this research, magnetic nanoparticles Fe3O4@Ca/MnCO3 were prepared and used to actively and efficiently deliver antigens to the cytoplasm of DCs, promote antigen cross‐presentation and DC activation, and finally enhance the cellular immune response of DC vaccines. The results show that the magnetic nanoparticles can actively and quickly deliver antigens to the cytoplasm of DCs by regulating the magnetic field, and achieve cross‐presentation of antigens. At the same time, the nanoparticles degradation product Mn2+ enhanced immune stimulation through the interferon gene stimulating protein (STING) pathway, and another degradation product Ca2+ ultimately promoted cellular immune response by increasing autophagy. The DC vaccine constructed with the magnetic nanoparticles can more effectively migrate to the lymph nodes, promote the proliferation of CD8+ T cells, prolong the time of immune memory, and produce higher antibody levels. Compared with traditional DC vaccines, cytoplasmic antigen delivery with the magnetic nanoparticles provides a new idea for the construction of novel DC vaccines.

effects. In this research, magnetic nanoparticles Fe 3 O 4 @Ca/MnCO 3 were prepared and used to actively and efficiently deliver antigens to the cytoplasm of DCs, promote antigen cross-presentation and DC activation, and finally enhance the cellular immune response of DC vaccines. The results show that the magnetic nanoparticles can actively and quickly deliver antigens to the cytoplasm of DCs by regulating the magnetic field, and achieve cross-presentation of antigens. At the same time, the nanoparticles degradation product Mn 2+ enhanced immune stimulation through the interferon gene stimulating protein (STING) pathway, and another degradation product Ca 2+ ultimately promoted cellular immune response by increasing autophagy.
The DC vaccine constructed with the magnetic nanoparticles can more effectively migrate to the lymph nodes, promote the proliferation of CD8 + T cells, prolong the time of immune memory, and produce higher antibody levels. Compared with traditional DC vaccines, cytoplasmic antigen delivery with the magnetic nanoparticles provides a new idea for the construction of novel DC vaccines. With the huge therapeutic potential, cancer immunotherapy is expected to become the mainstream of cancer treatment. Tumor vaccines, as an important immunotherapy method, have attracted wide attention due to their strong specificity, low side effects, generation of immune memory to long-term supervise the tumor metastasis, and recurrence. [1][2][3] Like traditional vaccines, ordinary tumor vaccines containing tumor antigens/adjuvants can be directly immunized to hosts. 4 However, it has been found that conventional tumor vaccines cannot be effectively taken up by antigen presenting cells (APCs) in the body, cannot effectively activate APCs, and fail to produce effective cellular immunity, resulting in poor therapeutic effects. [4][5][6] To solve this problem, researchers isolate the patient's own dendritic cells (DCs) out of the body and treated them directly in vitro with tumor antigen/adjuvant complex to promote the uptake of the tumor antigen by the DCs. The DCs sensitized in this way are called DC vaccines. Compared with ordinary tumor vaccines, these DC vaccines have more advantages. First, vaccination with the DC vaccines that are sensitized in vitro will greatly save the time for DCs to take up antigen and mature in the body, and activate T cells more quickly and effectively. Second, it is easier for the DC vaccines to activate CD8 + T cells. 7 Third, the safety of DC vaccines is higher. 8 At present, four DC tumor vaccines have been approved for marketing in the world. Among them, the DC vaccine (Provenge) was first approved by the Food and Drug Adminisrtation (FDA) for the treatment of prostate cancer in 2010. 9 Although many DC vaccines have entered clinical trials, the clinical response effectiveness of patients is still low. For example, the objective clinical response rate of the DC vaccines for prostate cancer and renal cell carcinoma is only 7.7%-12.7%. 10,11 The main reasons for the inefficiency of DC vaccines include: poor activation of DCs in vitro, reduced activity and migration ability of DCs due to long-term in vitro culture. As a result, the DC vaccines loaded with antigens usually stay at the injection site for a long time and are eliminated within 48 h. 12 15 Downregulation of the expression of cytokine signal transduction inhibitor 1 in DCs using siRNA can enhance the immunotherapy effect of DC vaccines. 16 Using the CD40 ligand expressed by T or B cells to bind to CD40 on the surface of DCs to increase expression of their costimulatory molecules can increase the efficacy of the DC vaccines. 17 Using physical techniques such as electroporation can promote the transfection of mRNA to DCs and enhance their antigen presentation. 18 Although these methods increase the anti-tumor immune response of DC vaccines to a certain extent, there are still problems to be solved, such as complicated preparation technology, low preparation success rate, high cost, and low response rate. Therefore, there is an urgent need to develop easily prepared, low-cost, efficient, and safe DC vaccines.
In recent years, magnetic nanoparticles have been widely used in tumor diagnosis, treatment, and imaging because of their good biocompatibility, stable performance, easy surface modification, and magnetic controlled drug delivery and release. [19][20][21][22] For example, Chiang et al. 22 used magnetic nanoparticles to load checkpoint inhibitor and T-cell activators, which could target to tumors by applying an external magnetic field to achieve in situ expansion of tumor-infiltrating T cells and repair the immunosuppressive tumor microenvironment. Inspired by the directional movement of magnetic nanoparticles, we propose to use magnetic nanoparticles to deliver tumor antigens to the DC cytoplasm under magnetic field, promote antigen cross-presentation, activate the DCs at the same time, and thereby enhance the cellular immune response. In addition, as reported, Mn 2+ can activate cyclic guanosine monophosphate adenosine monophosphate synthase-interferon gene stimulating protein (cGAS-STING) cascade reaction to induce antigen cross-presentation of DCs, 23,24 and Ca 2+ can regulate autophagy, which will help increase antigen cross-presentation. [25][26][27] 25,26 and also promote antigen cross-presentation. 27 The magnetic nanoparticles-based antigen delivery system provides a new strategy for the development of novel DC vaccines.

| Isolation and stimulation of bone marrow-derived dendritic cells
Bone marrow-derived dendritic cells (BMDCs) were obtained from the healthy female C57BL/6 mice. Briefly, the bone marrow cells were obtained from tibias and femurs, and red blood cells in it were lysed with red blood cell lysate. These bone marrow cells were cultured with RPMI1640 complete medium (containing 20 ng/ml GM-CSF and 10 ng/ml IL-4) and seeded into 6-well plates. The medium was replaced in every 2 days. On the 6th day, the immature BMDCs were seeded into 24-well low attachment surface plates (1 Â 10 5 cells/well) and treated

| Coculture assays
The above-activated BMDCs were cocultured with naive splenocytes in 24-well plates at a splenocytes/BMDCs ratio of 1,000,000/100,000.

| Immunohistochemical analysis
The female C57BL/6 mice (4-6 weeks old) were randomly divided into five groups (n = 5). Subsequently, the mice were subcutaneously immunized with 100 μl of the OVA-activated DCs (1 Â 10 6 cells/ mouse). On the 2nd and 7th day, the spleens and lymph nodes of the mice were collected, and fixed with 4% paraformaldehyde solution for further immunohistochemical analysis. The distributions of antigen protein OVA in spleens and lymph nodes were observed by an optical microscope (Leica DMI6000, Germany).

| Immunization evaluations of the DC vaccines in vivo
The C57BL/6 female mice (4-6 weeks old) were randomly divided into five groups (n = 5) and subcutaneously immunized with 100 μl of On the 7th day after the 2nd vaccination, the sera and splenocytes were separated from the mice.
In addition, the immunological evaluation of Fe 3 O 4 /OVA or

| Determination of OVA-specific antibodies in sera
The OVA-specific antibodies in the sera were detected with ELISA.
First, the antigen OVA solution (10 μg/ml) was prepared with carbonate buffer (0.1 M, pH = 9.6) and added to 96-well plates (100 μl/well) for coating overnight at 4 C. On the 2nd day, the 96-well plates were washed three times with PBS containing 0.05% Tween (PBST) and incubated at 37 C for 1 h with 200 μl of blocking solution (PBST solution containing 2% bovine serum albumin). Subsequently, the 96-well plates were washed three times and incubated for 2 h with 100 μl of diluted sera samples (dilution ratio: 1000). Then, the 96-well plates were washed with PBST three times and incubated at 37 C for 1 h with the horseradish peroxidase-conjugated goat anti-mouse IgG antibody solution (100 μl/well). Subsequently, the 96-well plates were washed with PBST four times, and incubated in dark for 15 min with the 3,3 0 ,5,5 0 -tetramethylbenzidine substrate solution (100 μl/well).
Finally, the 96-well plates were incubated with H 2 SO 4 solution (100 μl/well) to stop the enzymatic reaction. Then, the optical absorbances (ODs) of the wells were read at 450 nm by the microplate reader.

| Evaluation of histocompatibility of the DC vaccines
The five main tissues (heart, liver, spleen, lung, and kidney) of the mice were collected and fixed with 4% paraformaldehyde solution to prepare the tissue slices. Subsequently, those tissue slices were stained with hematoxylin-eosin staining solution and observed with the optical microscope.

| Statistical analysis
The obtained data were statistically analyzed using GraphPad Prism 5 software and the differences between the groups were analyzed using one-way ANOVA test. The data were expressed as the mean ± standard deviation (Mean ± SEM). *p < 0.05, **p < 0.01, and ***p < 0.001 were used to indicate the significant differences. As shown in Figure 1i, the OVA loading ability of the  [43][44][45][46][47][48][49] In this study, the OVA amount loaded on Fe 3 O 4 @Ca/MnCO 3 nanoparticles was 56 μg/mg, which has met the application of immunization.
Subsequently, the release of OVA was detected with BCA kit at different times, as shown in Figure S1D.

| Nanoparticles enhance the maturation of BMDCs and antigen presentation
Exogenous antigens with low immunogenicity and stability, 50 are usually presented to CD4 + T cells through the MHC class II molecules.
However, nanoparticles act as reservoirs of antigens, can promote antigen uptake, and present antigen to CD8 + T cells with MHC class I molecules to induce cellular immunity. 51,52 In addition, costimulatory molecules such as CD40, CD80, and CD86 are necessary for all antigen presentations.
In this study, the isolated BMDCs were treated with the mag- In addition, the levels of BMDC maturation and antigen presenta-

| Cellular uptake of the magnetic nanoparticles
Antigen uptakes are key steps for APCs' activation in the generation of potent immune responses. 53 The antigen delivery performance of be beneficial to antigen cross-presentation. 56 In this study, MDCs staining was used to label the autophagic vacuoles of DCs induced by and evaluated its function to stimulate CD8 + T cell proliferation and cytokine secretion in vitro. As shown in Figure 6a,b, The DCs activated by the Fe 3 O 4 @Ca/MnCO 3 containing nanoparticles could significantly increase the proportion of CD8 + T cells in T cells, which will contribute to cellular immunity.
Subsequently, the supernatant of the co-culture of BMDCs and splenocytes was tested. IFN-γ is an important cytokine to promote the production of antibody IgG2a and differentiation of CD8 + T cells into cytotoxic T lymphocyte (CTLs). 57 And TNF-α is also highly related to antitumor immunity. 58 The above results also indicate that the DC activated by the magnetic nanoparticles and magnetic field facilitated the delivery of antigens and promoted the migration of DCs to peripheral immune organs.

| DC vaccine induces an effective immune response in vivo
Antibody titer is a key indicator of the level of humoral and cellular immunity. The OVA-specific antibody titers in the sera of the The spleens contain a variety of immune cells, which play important roles in immune responses. 66 When exposed to the same antigen again, the cells will proliferate rapidly and the T cells will produce fast and effective immune responses. 67  response to re-exposed antigens.
Then, the proportion of CD8 + T cell in splenocytes and the cytokine secretion level was measured, as shown in Figure 10a Formation of immune memory T cells is the most important characteristic of vaccination, which plays a key role in immune surveillance by rapidly and potently responding to the re-exposed antigens. 68 Memory T cells can be divided into two subgroups, namely, effector memory T cells (T EM cells, CD44 high CD62L low ) and central memory T cells (T CM cells, CD44 high CD62L high ). 69 Here, the percentage of T EM cells among the splenocytes was detected. As shown in Figure 11

CONFLICT OF INTEREST
The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author.